Compositions of placentally-derived stem cells for the treatment of cancer

ABSTRACT

Disclosed are preparations of placentally-derived stem cells and compositions useful for the treatment of cancer. Said stem cells and compositions function through inducing a “guided differentiation” program in cancer cells, thereby reducing malignancy. Further extension of the invention pertains to augmenting ability of administered cells to induce differentiation through the co-administration of known differentiation inducing agents. Within the context of this disclosure, methods for inducing host responses to cancer are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 60/699,579, which was filed on Jul. 14,2005, the disclosure of which is hereby incorporated by reference in itsentirety.

BACKGROUND

1. Field of the Invention

The invention disclosed relates generally to the fields of cancertherapy. More particularly, it relates to the process of cancer celldifferentiation into cells of reduced malignancy, or lacking ofmalignancy. More specifically, the invention pertains to the utilizationof cord-blood and placentally derived stem cells for the stimulation ofcancer cell differentiation.

2. Description of Related Art

In the field of cellular biology, the definition of “stem cell” isdependent on function more than on morphology. The characteristicfunction of a stem cell is the ability to both self-renew and todifferentiate. Stem cells differentiate upon need of the host inresponse to a wide variety of stimuli, these include the need forincreased erythrocytes due to high altitude, or the need for augmentednumbers of neutrophils in response to a bacterial infection [1].Additionally, stem cells can be artificially induced to replicatethrough various means including administration of cytokines, growthfactors, and small peptides [2]. A wide variety of tissue-specific stemcells have been identified, these include liver [3], skin [4], renal[5], pancreatic [6], gastric [7] and neuronal [8].

There are many similarities between neoplastically transformed tissueand stem cells in the sense that both express embryonic-like features,including the ability to migrate (metastasize), ability to suppressimmune responses, and ability to proliferate. The sole differenceresides in the fact that stem cells are highly regulated in terms ofgrowth and differentiation, whereas cancer cells are not. At themolecular level, the more aggressive a cancer is, the morede-differentiated and “stem cell-like” it becomes. Specifically, regionsof DNA that are transcriptionally silenced in mature tissue, becomeactive when the tissue turns cancerous. For example the enzymetelomerase is needed for cells to escape proliferative senescence (iemultiple past the Hayflick limit). Stem cells possess active telomerase,in similarity to cancer cells. In contrast, as cells differentiate intospecialized tissue, telomerase expression becomes silencedepigenetically through synergistic involvement of DNA methylation andhistone deacetylation [9]. Conversely, tumor cells selectively silence,again through epigenetic mechanisms, genes that stop the tumor frombeing neoplastic. For example it is known that in prostate cancer tumorsuppressor genes become silenced by methylation [10]. In fact, this is acommon phenomena seen in a wide variety of histologically-differingtumors [11].

Stem cells actively secrete differentiation inducing factors, both knownand unknown, that are capable of inducing epigenetic reprogramming incells. In U.S. Patent Publication No. 2003/0211603 (Earp et al., herebyincorporated by reference in its entirety) disclose that embryonic stemcells possess the ability to reprogram differentiated cells to take a“de-differentiated” phenotype. Although the subject matter did notindicate potential application to cancer cells, such an applicationwould be counterintuitive since more dedifferentiated tumors would beexpected to possess increased malignancy. The invention disclosed inthis patent is based on the unique and unanticipated discovery that such“reprogramming” of cancer cells does not lead to increased malignancybut instead induces a differentiation program which results in formationof normal, or normal-like progenitors.

Cord blood is a rich source of hematopoietic stem cells, capable ofreconstituting the hematopoietic lineage in NOD-SCID mice. The potentialutilization of cord blood stem cells, such as CD34+ cells, has led tothe cryopreservation of such cells for more than a decade [12]. Indeed,the clinical use of cord blood derived stem cells is becoming increasingacceptable as an alternative to bone marrow transplant, or cytokinemobilized peripheral blood stem cell transplant for a wide variety ofdiseases. This is in part because of a reduced incidence of graft versushost reaction using cord blood stem cells in comparison with othersources [13]. Indeed, this was observed in pediatric leukemia patientslacking suitable major histocompatibility complex (MHC) matched donors[14], as well as in transplantation of patients with certain anemias orhematopoietic stem cell disorders such as beta-thalassemia major [15].

Placental extracts have also been previously described in the art. Forexample, a low molecular weight fraction of placental tissue is known tobe immunosuppressive as reported by Chauaot. Additionally, the use ofplacenta in cosmetics is well known. Therefore, placenta and cord-bloodare two accepted sources of biomaterial that is innocuous for human use.

SUMMARY OF THE INVENTION

The invention described in the present disclosure teaches methods ofgenerating compositions therapeutically useful for treatment of cancerpatients. While chemotherapy and radiation therapy induce severe sideeffects through collateral damage to non-cancerous cells and organs, thepresent invention utilizes the ability of cancer cells to be“reprogrammed” into benign, non-cancerous progeny. The use of cellulardifferentiation therapy has been previous attempted in cancer patients;however, the majority of such differentiation inducers have beensynthetic chemicals administered at unnaturally high doses.

In the present invention, the inventor teaches the utilization of stemcells to guide the differentiation of cancer cells into non-malignantcells. For example, it is disclosed that the administration of cordblood stem cells to a cancer patient is demonstrated to have someanticancer effect. This effect is magnified if certain derivatives fromthe cord blood stem cells are systemically administered. Saidderivatives include the low molecular weight fraction that can bepurified through column chromatography. Other methods of separationinclude high performance liquid chromatography, Fas Q liquidchromatography, or on-line mass spectrometry.

A simpler method of concentrating the differentiation inducing abilitiesof the cord blood stem cells is through purified the cell culturesupernatant of said cells through solid phase adsorption such as throughthe use of a C-18 columns. The therapeutic composition can also beproduced in a more convenient manner through utilization of placentalysate concentrated by C-18.

Noted within the disclosure is that cancer cells treated withdifferentiation inducing agents possess an inhibited proliferativeindex. This is attributed to the fact that the differentiation inducingcomposition endows the neoplastic cells with a more benign phenotype(i.e. reduced proliferation).

The present disclosure is not intended to be limiting in any way and canbe practiced with other techniques known to one skilled in the art. Forexample, co-administration of placental extract with differentiationinducers known in the art is within the scope of the invention.

Additionally, it is known that differentiated cells express antigens notfound in immature progeny. U.S. Patent Publication No. 2004/0185563,hereby incorporated by reference in its entirety, describes a method ofincreasing efficacy of immunotherapy by induction of neoantigens oncancer cells through differentiation by administration of histonedeacetylase inhibitors. An application of the invention disclosedincludes the utilization of stem cell derivatives for increasing theimmunogenicity of tumor tissue. This would result in sensitization toconventionally used immune stimulatory anticancer approaches includingbut not limited to cancer vaccines, cytokine therapy, and non specificimmune stimulators such as BCG, CpG, or beta glucan.

In some embodiments, a composition comprising placentally-derived tissueextract and cells is provided, which is capable of inducing thedifferentiation of a substantially neoplastic cell into a cell withbenign characteristics. The placental tissue can be human. Theneoplastic cell can possess one or more of the followingcharacteristics: invasiveness, ability to metastasize, ability tosuppress immune response, ability to proliferate past the Hayflicklimit. The neoplastic cell can be chosen from, for example, the group ofcancers consisting of: a soft tissue sarcoma, a lymphoma, a cancer ofthe brain, an esophageal cancer, a uterine cancer, a cancer of thecervix, a bone cancer, a lung cancer, a cancer of the endometrium, abladder cancer, a breast cancer, a cancer of the larynx, a cancer of thecolon/rectum, a stomach cancer, a cancer of the ovary, a pancreaticcancers, an adrenal gland cancer and a prostate cancer. The cell withbenign characteristics can be, for example, a cell that lacks one ormore of the following: invasiveness, ability to metastasize, ability tosuppress immune response, and ability to proliferate past the Hayflicklimit. The placental tissue may be processed, for example, throughcollagenase or other cell dissociating methods, followed by purificationof single cells. For example, the cellular purification can be performedusing Percoll gradient of 0.3 mg/ml or other density gradients. Theplacentally-derived cells can be, for example, cord blood stem cells,mesenchymal stem cells, or other placentally derived stem cells. Theplacentally-derived cells and tissue extract can be administered, forexample, to a patient in need thereof together with a compound capableof inducing cellular differentiation. The compound capable of inducingcellular differentiation can be, for example, dihydroxyvitamin D3,retinoic acid, valproic acid, or a histone deacetylase inhibitor. Thehistone deacetylase inhibitor can be, for example, trichostatin A, ashort-chain fatty acid, a hydroxamate, a cyclic tetrapeptide, or abenzamide. The composition can be used as an adjuvant to achemotherapeutic drug.

In additional embodiments, a method of reducing malignancy of a tumor ina patient is provided, by extracting placental tissue obtained from ahealthy mother, homogenizing said tissue and obtaining a single cellsuspension, purifying CD34+ cells, and administering the (CD34+ cells toa patient in need thereof. The CD34+ cells can be, for example,co-administered with a differentiation-inducing agent. The CD34+ cellscan be, for example, expanded in vitro prior to administration. Theexpansion can be performed by culture in media conditioned withcytokines and/or growth factors selected from the following: leukemiainhibitory factor, IL-1 through IL-13, IL-15 through IL-17, IL-19through IL-22, granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolon) stimulating factor (M-CSF), erythropoietin (Epo), thrombopoietin(Tpo), Flt3-ligand, B cell activating factor, artemin, bone morphogenicprotein factors, epidermal growth factor (EGF), glial derivedneurotrophic factor, lymphotactin, macrophage inflammatory proteins,myostatin, neurturin, nerve growth factors, platelet derived growthfactors, placental growth factor, pleiotrophin, stem cell factor, stemcell growth factors, transforming growth factors, tumor necrosisfactors, Vascular Endothelial Cell Growth Factors, and fibroblast growthfactors, FGF-acidic and basic fibroblast growth factor. In additionalembodiments, one or more genes can be transfected into the CD34+ cellsprior to administration. The transfected genes can be associated withinhibition of tumor growth. The transfected genes can be selected fromthe group consisting of: IFN-g, IL-2, IL12, IL-15, IL-18, IL-23.

A chemotherapeutic agent can also be co-administered with the CD34+cells. The chemotherapeutic agent can be chosen from, for example,methotrexate, vincristine, adriamycin, cisplatin, non-sugar containingchloroethyinitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MM1270, BAY 12-9566, RAS famesyltransferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514,LY264618/Lometexol, Glamolec, Cl-994, TNP-470, Hycamtin/Topotecan,PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin,Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710,VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat,CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,Temodal/Temozolomide, Evacet/liposomal doxorubicin,Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) and Vindesine sulfate.

In an additional embodiment, a cell free extract capable of inducingdifferentation in cancer cells is provided, which is produced byextracting human placenta; homogenizing said placenta, resulting in ahomogenate; centrifuging said homogenate in order to free thesupernatant of cellular debris; purifying differentiation associatedmolecular weight fractions; and administering said fractions to apatient in need thereof. The purified, differentiation-inducing fractioncan have, for example, a molecular weight of 10-10,000 Daltons, or amolecular weight of 300-3,000 Daltons. The cell-free extract can be usedin a method of stimulating an immune response to cancer cells byadministration of the extract, together with an appropriateimmunological adjuvant. In additional embodiments, a method ofprophylactically treating a cancer patient is provided by administrationof the cell free extract a patient in need thereof. In an additionalembodiment, a method of inhibiting progression of a preneoplastic lesionto neoplasia is provided, by administration of the cell free extract.The cell free extract can, for example, be derived from bone marrow stemcells or unpurified bone marrow nucleated cells as an alternative tocord blood or other placentally-derived cells.

In an additional embodiment, a differentiation inducing composition,suitable for administration to a patient in need thereof, and derivedfrom placental tissue is provided, by extracting human placenta,homogenizing the placenta, resulting in a homogenate, centrifuging thehomogenate in order to free the supernatant of cellular debris,purifying the cell free-debris through adsorption on a C-18 column; andfilter sterilizing the composition. The homogenized portion of theplacenta may consist substantially of, for example, chorionic tissue. Ahistone deacetylase inhibitor can be added directly to the composition.In additional embodiments, a the composition can be administeredintratumorally in a method of inducing differentiation and reprogrammingof distal cancer lesions. In additional embodiments, a method ofsubstantially increasing efficacy of the composition is provided, byadministration in a liposomal complex. A substantially purifiedtherapeutic composition can be prepared, for example, by preparing asupernatant, subjecting the supernatant to solid phase extraction toproduce a product, subjecting the resulting product to gel filtration toproduce a product, subjecting the resulting product to anion exchangefast phase liquid chromatography to produce a product, subjecting theresulting product to amino-high performance liquid chromatography toproduce a product, and subjecting the resulting product to reverse phasehigh performance liquid chromatography to produce purifieddifferentiation inducing factor. The supernatant can be prepared, forexample, by culturing cord-blood derived stem cells to produce afactor-containing supernatant, or by centrifugation of lysed placentalextract.

DESCRIPTION OF FIGURES

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a bar graph demonstrating the induction of HL-60differentiation in vitro by co-culture with cord blood nucleated cells.

FIG. 2 is a bar graph demonstrating the induction of HL-60differentiation in vitro by co-culture with cord blood CD34+ stem cells.

FIG. 3 is a bar graph demonstrating the induction of HL-60differentiation by culture with placental extracts.

FIG. 4 is a bar graph demonstrating the induction of HL60differentiation using cord blood extracts.

FIG. 5 is a bar graph demonstrating the inhibition of HL-60proliferation by culture with placental extracts.

FIG. 6 is a bar graph demonstrating the inhibition of LNCaPproliferation by culture with placental extracts.

FIG. 7 is a bar graph demonstrating the inhibition of PC-3 proliferationby culture with placental extracts.

FIG. 8 is a bar graph demonstrating the inhibition of Lewis LungCarcinoma cell proliferation by culture with placental extracts.

FIG. 9 is a bar graph demonstrating the inhibition of lung metastasisusing placental extract in the Lewis Lung Carcinoma murine model of lungcancer.

FIG. 10 is a table showing the inhibition of Lewis Lung Carcinoma growthusing placental extract.

DETAILED DESCRIPTION OF THE INVENTION

Without intending to be limited by theory, it is also hypothesized thatfactors present in the culture of cord blood stem cells have the abilityto induce a differentiation program in cancer cells in effect, to begina reprogramming process that endows the cell an ability to make progenywith benign, noncancerous, or semicancerous phenotype. Simultaneously orsubsequently, the cell encounters growth conditions or agents thatpromote differentiation down another differentiation pathway. Theseideas are placed here for the interest of the skilled artisan, and donot need to be understood to put the invention into practice. Theclaimed invention is not restricted by mechanism of action, and islimited only by the features explicitly stated, as interpreted by oneskilled in the art.

The invention described teaches methods of inducing the differentiationof cancer cells in a host in need thereof through administration ofcells or factors derived from said cells that possess stem cell likeproperties. Specifically, the differentiation-inducing capabilities ofthe compositions described are useful for the treatment of breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor,primary brain tumor, acute and chronic lymphocytic and granulocytictumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma,pheochromocytoma, mucosal neuronms, intestinal ganglloneuromas,hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor,seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and insitu carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma,malignant carcinoid, topical skin lesion, mycosis fungoide,rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma,malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, malignant melanomas, andepidermoid carcinomas.

As described herein, cells with stem cell activity, such as cord bloodcells, can be derived from human placental tissue. Thus, the terms“placentally derived tissues” and “placentally derived cells” caninclude cord blood cells.

In some embodiments, cells with stem cell like activity are purifiedfrom human placenta using antibody mediated cell depletion followed bypositive selection. Methods of depleting lineage positive cells are wellknown in the art and include the use of antibodies to the lineageantigens (CD5, CD45R (B220), CD11b, anti-Ly-6G (Gr-1), 7-4, and Ter-119)followed by complement depletion. A less damaging protocol involvesutilization of magnetically bound antibodies and depletion by use ofmagnetic separation. This protocol is well known in the art and iscommercially available using the Magnetic Activated Cell Sorting (MACS)technology offered by Miltenyi Biotech. Further purification of cellswith stem cell activity can be performed by selection for the stem cellantigen CD34. Selection can be accomplished again by magnetic separationusing the “positive selection” method, or alternatively by fluorescentactivated cell sorting (FACS). Other methods of purifying cells withstem cell activity include use of Wheat Germ Agglutinin, or selectionfor low density profile using a chemical gradient such as Percoll.

Methods for expansion of stem cell populations have been described. Forexample, U.S. patent application Ser. No. 11/353,692 filed on Feb. 14,2006, entitled METHOD FOR EXPANSION OF STEM CELLS, describes a method ofincreasing the growth of stem cells by mixing the stem cells with agrowth medium that has been conditioned by an incubation with placentaltissue. This method increases the expansion of the stem cell population.This patent application is incorporated by reference herein in itsentirety.

Cells with stem cell activity are recognized by ability to reconstitutesevere combined immunodeficiency mice with multilineage human bloodpopulations as determined by the species specific marker CD45. Othermethods of assessing the “stem cell” ability of purified cord bloodcells includes plating said cells in methylcellulose in the presence ofdifferentiation-inducing factors and assessing for production ofmultilineage colonies. For example, plating in the presence of G-CSFwould induce formation of granulocytes, plating in the presence of EPOwould induce erythrocyte formation, and plating in the presence ofthrombopoietic would induce colonies consisting of megakaryocytes.

Upon purification of a stem cell population, the cells can beadministered directly into a cancer patient through parenteralintroduction, comprising intramuscular, intravenous, peritoneal,subcutaneous, intradermal, as well as topical administration. Apreferred method of administration is intravenous, however, variouscompositions of the cells can be derived by one skilled in the art. Forexample, when treating a patient with melanoma, the purified cord bloodstem cells can be lyzed and admixed with transdermal carrying agents foreffective direct delivery. Transdermal carriers include systems such asiontophoretic and sonophoretic systems, thermosetting gels, andprodrugs. For enhanced uptake, absorption promoters may be utilized. Theabsorption promoters may be selected in particular, from propyleneglycol, hexylene glycol, propylene glycol dipelargonate, glycerylmonoethyl ether, diethylene glycol, monoglycerides, monooleate ofethoxylated glycerides (with 8 to 10 ethylene oxide units), Azone(1-dodecylazacycloheptan-2-one), 2-(n-nonyl)-1,3-dioxolane,isopropylmyristate, octylmyristate, dodecyl-myristate, myristyl alcohol,lauryl alcohol, lauric acid, lauryl lactate, terpinol, 1-menthol,d-limonene, β-cyclodextrin and its derivatives or surfactants such aspolysorbates, sorbitan esters, sucrose esters, fatty acids, bile salts,or alternatively lipophilic and/or hydrophilic and/or amphiphilicproducts such as poly-glycerol esters, N-methylpyrrolidone,polyglycosylated glycerides and cetyl lactate. The absorption promoterpreferably represents from 5 to 25% of the weight of the composition.Further description of absorption promoters appears in U.S. Pat. No.6,538,039, which is hereby incorporated by reference in its entirety.

In terms of administrating the cells through the intravenous route,several modifications can be made to the cells to increase efficacy,this includes transfection with suicide genes, immune stimulatory genes,or genes which encode pro-drug activating enzymes. Additionally, thecord blood stem cells may be labeled in order to detect presence oftumor cells.

Typical administration of cells is performed based on the therapeuticneed of the patient. In a standard embodiment, cord blood stem cells areinfused intravenously in a physiologically suitable solution such asalbumin with saline. Cell concentrations may range from 10³-10⁹ CD34+cells/kg. Frequency may include weekly, daily, or depending on status oftumor growth as determined by biomarkers, imaging or patient generalmedical condition.

Another embodiment deals with using placental extracts as aheterogeneous starting point for preparation of a pharmaceutical agent.This embodiment uses full term, healthy, pathogen free chorions obtainedfrom deliveries or cesarean sections deliveries. Many methods exist forpreparation of cell lysate. Cell lysis procedures were extensivelydescribed in US Patent Publication No. 2003/0211603, hereby incorporatedby reference in its entirety. Procedures described therein are modifiedfor cord blood stem cells. For example, cell lysate or extract can beprepared from cord blood stem cell cultures or bone marrow stem cellcultures. Before lysing, the cells typically are allowed to recoverafter the last passage by culturing for about 2-3 days in standardmedium until the culture is at least about 50% confluent. The cells canbe lysed directly in the culture dish, for example, by replacing themedium with a solubilizing liquid, or by repeated freeze-thawing.Alternatively, the cells can be resuspended from the culture surfacebefore lysis, for example, by brief collagenase digestion, or byscraping. The resuspended cells are collected, for example, bycentrifugation, and then lysed by adding a suitable solvent, byfreeze-thawing, by shearing through a narrow-gauge needle or in a tissuegrinder, by sonicating, by mechanical homogenization, or by any othersuitable method. In certain circumstances, subcellular organelles can beremoved or enriched, or membrane fractions can be prepared, according tostandard methods. Techniques in subfractionating cells to produce cellcomponents and extracts can be found in Storrie et al., Meth. Enzymol.182:203, 1990; and in Subcellular Fractionation: A Practical Approach(Grahan & Rickwood, eds., Oxford, 1997), both of which are herebyincorporated by reference in their entirety. Viscosity of the cellextract caused by long-chain nucleic acids can be reduced by treatingwith DNAse, or other appropriate nucleases. Non-ionic detergents with ahigh critical micelle concentration (such as sodium deoxycholate) can beremoved by dialysis. Other detergents (such as Triton™ X-100, octylglucoside, or Nonidet™-P40) can be removed, for example, on adsorbentbeads or chromatography columns. Large particulates can be removed, forexample, by centrifugation or microfiltration. As another option, highmolecular weight solutes can be concentrated from the clarified extract(for example, by microfiltration, salt precipitation, columnchromatography, or lyophilization).

In accordance with a further embodiment, a method is provided forpreparing a purified cancer differentiation inducing factor comprisingthe steps of

(a) culturing cord-blood derived stem cells to produce afactor-containing supernatant;

(b) subjecting the supernatant to solid phase extraction to produce aproduct;

(c) subjecting the product from step (b) to gel filtration to produce aproduct;

(d) subjecting the product from step (c) to anion exchange fast phaseliquid chromatography to produce a product;

(e) subjecting the product from step (d) to amino-high performanceliquid chromatography to produce a product; and

(f) subjecting the product from step (e) to reverse phase highperformance liquid chromatography to product purified differentiationinducing factor.

Bone marrow, either fetal or adult, human, or from other animals mayalso be used as a source of stem cells for purification of thedifferentiation inducing composition. For example, human fetal bones maybe dissected from 21- to 24-week-old fetuses obtained by electiveabortion with approved consent (i.e., from Anatomic Gift Foundation,White Oak, Ga.). To purify human HSCs, BM cell suspensions are preparedby flushing split long bones with RPMI 1640 containing 2%heat-inactivated FCS (Gemini Bio-Products, Inc, Calabasas, Calif.).Low-density (<1.077 g/mL) mononuclear cells are isolated (Lymphoprep;Nycomed Pharma, Oslo, Norway) and washed twice in staining buffer (SB)consisting of Hanks'balanced salt solution (HBSS) with 2%heat-inactivated FCS and 10 mmol/L HEPES. Samples are then incubated for10 minutes with 1 mg/mL heat-inactivated human gammaglobulin (Gamimune;Miles Inc, Elkhart, Ind.) to block Fe receptor binding of mouseantibodies. Fluorescein isothiocyanate (FITC)-labeled CD34 monoclonalantibodies (MoAbs) and phycoerythrin (PE)-labeled thy-1 MoAbs are thenadded at 0.5 to 1 μg/10⁶ cells in 0.1 to 0.3 mL SB for 20 minutes onice. Control samples are incubated in a cocktail of FITC-labeled andPE-labeled isotype-matched MoAbs. Cells are washed twice in SB,resuspended in SB containing 1 μg/mL propidium iodide (Molecular ProbesInc, Eugene, Oreg.), and sorted using the tri-laserfluorescence-activated cell sorter (FACS) MoFlo (Cytomation, Inc, FortCollins, Colo.). Live cells (ie, those excluding propidium iodide) arealways greater than 95%. Sort gates are set based on the meanfluorescence intensity of the isotype control sample. Cells arecollected in 12- or 24-well plates in RPMI 1640 containing 10% FCS and10 mmol/L HEPES, counted, and reanalyzed for purity in every experiment.Typically, 450,000 to 500,000 CD34+ thy-1⁺ cells are obtained from asingle donor. MoAbs for CD34 are purchased from Becton Dickinson(Mountain View, Calif.). MoAbs for thy-1 and isotype controls arepurchased from Pharmingen (San Diego, Calif.).

Using both the placental source described above or the bone marrowsource of differentiation-inducing activity, concentration of thisactivity can be performed using solid phase extraction. C₁₈ cartridges(Mini-Spe-ed C18-14%, S.P.E. Limited, Concord ON) are prepared bywashing with 10 ml of methanol followed by 10 ml 18 megaohm/cmdeionized-distilled water. Up to 100 ml of supernatants of cultured cordblood stein cells are passed through each cartridge before elution.After washing the cartridges with 5 ml of deionized-distilled water,material adsorbed to the C₁₈ cartridge is eluted with 3 ml methanol,evaporated under a stream of nitrogen, redissolved in a small volume ofmethanol, and stored at 4° C. Before testing the eluate fordifferentiation inducing activity in vitro, the methanol is evaporatedunder nitrogen and replaced by culture medium.

C₁₈ cartridges can be used to adsorb small hydrophobic molecules fromthe cord blood stem cell culture supernatant, allowing the eliminationof salts and other polar contaminants.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

1. CD34+ Cell Purification

Cord blood was obtained from consenting mothers undergoing cesareandelivery of healthy, full-term infants, and low-density (less than 1.077g/ml) cells were isolated by centrifugation on Ficoll-Hypaque. CD34⁺cell-enriched populations (65-98% CD34⁺ cells) were obtained by removalof lineage marker-positive cells using a column (n=2); and positive(EasySep) selection using magnetic beads (n=1; StemCell TechnologiesInc.). Cells were stimulated overnight for in vivo experiments and for48 hours for in vitro experiments at densities less than or equal to2×10⁵ cells/ml in Iscove's medium supplemented with 1% BSA, 10 μg/mlbovine pancreatic insulin, and 200 μg/ml human transferrin (BIT;StemCell Technologies Inc.), 10⁻⁴ mol 2-mercaptoethanol, 2 mM glutamine,100 ng/ml FL (Immunex Corp.), 100 ng/ml SF, 50 ng/ml Tpo (GenentechInc.), and 100 ng/ml hIL-6 (provided by S. Rose-John,Christian-Albrechts University, Kiel, Germany). The following day, thecells were pelleted, resuspended in fresh growth factor-supplementedmedium with 5 μg/ml protamine sulfate and 0.5×10⁸ to 5×10⁸ infectiousunits/ml (MOI=9-140; 140 in experiment 1, 9 and 90 in experiment 2),placed in a 24-well plate coated with 2 μg/cm² Retronectin (Takara ShuzoCo.) or with 5 μg/cm² fibronectin (Sigma-Aldrich), and then incubated at37° C. for 6 hours.

2. Generation of Placental Lysate

Term placentas were harvested and the chorion was mechanically separatedunder sterile conditions. Placentas were collected in ice-cold Dutchmodification of RPMI 1640 (Sigma, St. Louis, Mo.) supplemented with 10%FCS, 2 mM L-glutamine, gentamicin (25 μg/ml), andpenicillin/streptomycin (100 U/ml) (complete medium). They wereincubated with occasional agitation for 20 min at room temperature incalcium- and magnesium-free HBSS (Life Technologies, Paisley, U.K.)containing 1 mM DTT (Sigma). Purified cell homogenate was prepared bycollagenase digestion. Briefly, bacterial collagenase (AdvanceBiofactures, Lynbrook, N.Y.) digestion was sequentially performed, 4 μlof 50 mM calcium acetate was added, followed by 3 μl (3 units) ofbacterial collagenase. Reactions were then incubated for another 60 minat 37° C. Heparitinase digestions (15 μl) were carried out for 90 min at37° C. in a pH 7.0 buffer consisting of 100 mM sodium acetate, 10 mMcalcium acetate, using 5 milliunits of enzyme. In those reactions wherebacterial collagenase digestion was sequentially performed, 4 μl of 5×collagenase buffer (250 mM Tris-HCl, pH 7.2, 50 mM calcium acetate) wasadded, followed by 3 μl (3 units) of bacterial collagenase. Reactionswere then incubated for another 60 min at 37° C. Purified cells weresubsequently sonicated, centrifuged at 10,000 g for 3 hours, andsupernatant was collected, filter sterilized, and quantified for proteincontent using the Bradfort Assay.

3. Induction of HL-60 Differentiation in vitro Using Cord BloodCo-Culture

As demonstrated in FIG. 1, coculture of 100,000 HL-60 humanmyelomonocytic leukemia cells with increasing numbers of cord bloodnucleated cells resulted in an increased proportion of HL-60 cellsdifferentiating into adherent monocytic cells. HL-60 cells were platedin 96 well plates at a concentration of 100,000 cells per well. Theaddition of cord blood nucleated cells was performed at theconcentration indicated in the figure. The percentage of adherent cellswas determined by a blinded observer. The fact that the HL-60 were trulydifferentiating was attested to by morphological changes (data notshown).

4. Induction of HL-60 Differentiation in vitro Using Purified Cord BloodCD34+ Cells

As demonstrated in FIG. 2, coculture of 100,000 HL-60 humanmyelomonocytic leukemia cells with increasing numbers of CD34+ purifiedcord blood nucleated cells resulted in an increased proportion of HL-60cells differentiating into adherent monocytic cells. HL-60 cells wereplated in 96 well plates at a concentration of 100,000 cells per well.The addition of cord blood CD34+ cells was performed at theconcentration indicated in the figure. The percentage of adherent cellswas determined by a blinded observer. The fact that the HL-60 were trulydifferentiating was attested to by morphological changes (data notshown).

5. Induction of HL-60 Differentiation by Culture with Cord BloodExtracts

Cord blood extracts were prepared by culturing nucleated cells in Hanksmedia for 48 hours and the supernatant was concentrated using SolidPhase Extraction. C₁₈ cartridges (Mini-Spe-ed C18-14%, S.P.E. Limited,Concord ON) were prepared by washing with 10 ml of methanol followed by10 ml 18 megaohm-cm deionized-distilled water. Up to 100 ml ofsupernatants of cultured cord blood stem cells are passed through eachcartridge before elution. After washing the cartridges with 5 ml ofdeionized-distilled water, material adsorbed to the C₁₈ cartridge iseluted with 3 ml methanol, evaporated under a stream of nitrogen,redissolved in a small volume of methanol, and stored at 4° C. Beforetesting the eluate for differentiation inducing activity in vitro, themethanol is evaporated under nitrogen and replaced by culture medium.Before addition to the media, however, the concentration of C₁₈ extractwas quantified using the Bradfort assay. The indicated concentration ofcord blood extract was added to 100,000 proliferating HL-60 cells perwell. As shown in FIG. 3, and increased number of differentiated HL-60cells was observed after 48 hours.

6. Induction of HL-60 Differentiation by Placental Extracts

Placental extracts were derived and quantified as described above. Theindicated concentration of cord blood extract was added to 100,000proliferating HL-60 cells per well. As shown in FIG. 4, an increasednumber of differentiated HL-60 cells was observed after 48 hours. Theextent of differentiation was dependent on the concentration ofplacental extract added.

7. Inhibition of Cancer Cell Proliferation by Culture with PlacentalExtracts

HL-60, LNCaP, PC-3 and Lewis Lung carcinoma cells (1×10⁵/well) wereplated in 96 well plates in 200 μl of RPMI 1640 (Life Technologies)supplemented with 10% FCS (Life Technologies), 100 U/ml of penicillin(Life Technologies), and 100 μg/ml of streptomycin (Life Technologies).Cells were cultured at 37° C. in a humidified atmosphere of 5% CO₂ for 3days in the presence of the indicated about of placental extract, andpulsed with 1 μCi of [³H]thymidine (Amersham Pharmacia Biotech) for thelast 16 h of culture. Cells were harvested onto glass fiber filters, andthe radioactivity incorporated was quantitated using a Wallac Betaplateliquid scintillation counter. Results were expressed as the mean cpm oftriplicate cultures±SEM. As seen in FIGS. 5-8, a dose-dependentinhibition of tumor cell proliferation was noted in all 3 in vitroexperimental tumor systems.

8. Inhibition of Cancer Cell Metastasis by Placental Extract

Lewis lung carcinoma (ATCC) were propagated by sequential subcutaneoustransplantation in C57BL/6 mice. A single-cell suspension of tumor cellswas prepared by mincing the tumor, followed by passage of the suspensionfirst through a stainless steel mesh and then through a series ofhypodermic needles of increasing gauge. This suspension of cells wasaliquoted and frozen. For each series of injections, an aliquot offrozen tumor cells was thawed and expanded by culturing for 3 passagesin Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum, penicillin, and streptomycin. Be fore injection, the tumor cellswere detached by trypsinization, washed once in cold DMEM containing 10%fetal calf serum, and once in cold serum-free medium. Cell viability wasusually greater than 85%, as determined by trypan blue exclusion. Thedorsal skin of mice was shaved 1 day before injection. The mice wereanesthetized by Metofane inhalation, and tumor cells (500,000) trypanblue-excluding cells in 100 μL) were injected into the dorsal subcutisusing a 27-gauge needle.

Three weeks after tumor inoculation quantitation of lung metastasis wasperformed. Mice were killed using CO2 narcosis, and the lungs wereremoved, rinsed in phosphate-buffered saline (PBS), and placed inBouin's fixative for at least 24 hours. The fixed lungs were carefullyseparated into individual lobes with forceps, and the number of surfacemetastases (appearing as white foci against a yellow background) wascounted for each lobe using a dissecting microscope at 4×magnification(total metastatic foci) or by the naked eye (large metastatic foci).

Two treatment protocols were used. In the first one, lug of placentalextract was administered 1 day prior to tumor cell inoculation, in thesecond protocol 1 ug of placental extract was administered on days −1,1, 4, and 7 in reference to tumor cell inoculation. As seen in FIG. 9,protocol 2 was most effective at decreasing the amount of visible tumorfoci.

9. Inhibition of Tumor Growth by Placental Extract

C57/BL6 mice were inoculated with Lewis Lung Carcinoma cells asdescribed in the above section. Tumor growth was quantitated by ablinded observer using calipers. The size of the tumor is represented inFIG. 10. As observed, the mice treated with Protocol 2 (described above)resulted in a significant inhibition of tumor growth. Although notillustrated, the survivors had a dense, fibrous scar-like tissue wherethe tumor used to be.

It will be apparent to one skilled in the art that varying substitutionsand modifications can be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

Those skilled in the art recognize that the aspects and embodiments setforth herein can may be practiced separate from each other or inconjunction with each other. Therefore, combinations of separateembodiments are within the scope as disclosed herein.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions indicates the exclusion ofequivalents of the features shown and described or portions thereof. Itis recognized that various modifications are possible within the scopeof the invention disclosed. Thus, it should be understood that althoughthe present invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the disclosure.

The following references are incorporated herein by reference in theirentireties.

REFERENCES

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1. A composition comprising placentally-derived tissue extract andcells, capable of inducing the differentiation of a substantiallyneoplastic cell into a cell with benign characteristics.
 2. Thecomposition of claim 1, wherein the placental tissue is human.
 3. Thecomposition of claim 1, wherein said neoplastic cell possesses one ormore of the characteristics selected from the following: invasiveness,ability to metastasize, ability to suppress immune response, ability toproliferate past the Hayflick limit.
 4. The composition of claim 3,wherein said neoplastic cell is selected from the group of a cancerconsisting of: a soft tissue sarcoma, a lymphoma, a cancer of the brain,an esophageal cancer, a uterine cancer, a cancer of the cervix, a bonecancer, a lung cancer, a cancer of the endometrium, a bladder cancer, abreast cancer, a cancer of the larynx, a cancer of the colon/rectum, astomach cancer, a cancer of the ovary, a pancreatic cancers, an adrenalgland cancer and a prostate cancer.
 5. The composition of claim 1,wherein said cell with benign characteristics lacks one or more of thefollowing: invasiveness, ability to metastasize, ability to suppressimmune response, and ability to proliferate past the Hayflick limit. 6.The composition of claim 1, wherein the placental tissue is processedthrough collagenase or other cell dissociating methods, followed bypurification of single cells.
 7. The composition of claim 6, whereinsaid purification of single cells is performed using Percoll gradient of0.3 mg/ml or other density gradients.
 8. The composition of claim 1,wherein said placentally-derived cells are cord blood stem cells.
 9. Thecomposition of claim 1, further comprising a compound capable ofinducing cellular differentiation.
 10. The composition of claim 9,wherein said compound capable of inducing cellular differentiation isselected from the group consisting of: dihydroxyvitamin D3, retinoicacid, or valproic acid.
 11. The composition of claim 9, wherein saidcompound capable of inducing cellular differentiation is a histonedeacetylase inhibitor.
 12. The composition of claim 11, wherein saidhistone deacetylase inhibitor is selected from the group consisting of:trichostatin A, a short-chain fatty acid, a hydroxamate, a cyclictetrapeptide, or a benzamide.
 13. The composition of claim 1, whereinsaid composition is an adjuvant to a chemotherapeutic drug.
 14. A methodof reducing malignancy of a tumor in a patient comprising: a. extractingplacental tissue obtained from a healthy mother; b. homogenizing saidtissue and obtaining a single cell suspension; c. purifying CD34+ cells;and d. administering the CD34+ cells to a patient in need thereof. 15.The method of claim 14, wherein a differentiation inducing agent isco-administered with the CD34+ cells.
 16. The method of claim 14,wherein said CD34+ cells are expanded in vitro prior to administration.17. The method of claim 16, wherein expansion is performed by culture inmedia conditioned with cytokines and/or growth factors selected from thegroup consisting of: leukemia inhibitory factor, IL-1 through IL-13,IL-15 through IL-17, IL-19 through IL-22, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin(Epo), thrombopoietin (Tpo), Flt3-ligand, B cell activating factor,artemin, bone morphogenic protein factors, epidermal growth factor(EGF), glial derived neurotrophic factor, lymphotactin, macrophageinflammatory proteins, myostatin, neurturin, nerve growth factors,platelet derived growth factors, placental growth factor, pleiotrophin,stern cell factor, stem cell growth factors, transforming growthfactors, tumor necrosis factors, Vascular Endothelial Cell GrowthFactors, and fibroblast growth factors, FGF-acidic and basic fibroblastgrowth factor.
 18. The method of claim 14, wherein one or more genes istransfected into the CD34+ cells prior to administration.
 19. The methodof claim 18, wherein said one or more transfected genes are associatedwith inhibition of tumor growth.
 20. The method of claim 19, whereinsaid one or more transfected genes are selected from the groupconsisting of: IFN-g, IL-2, IL12, IL-15, IL-18, IL-23.
 21. The method ofclaim 14, wherein a chemotherapeutic agent is co-administered with theCD34+ cells.
 22. The method of claim 21, wherein said chemotherapeuticagent is selected from the group consisting of: methotrexate,vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MM1270, BAY 12-9566, RAS famesyltransferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514,LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan,PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin,Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710,VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat,CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,Temodal/Temozolomide, Evacet/liposomal doxorubicin,Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) and Vindesine sulfate.
 23. A cell free extract capable ofinducing differentation in cancer cells, the cell free extract producedby the process comprising: a. extracting human placenta; b. homogenizingsaid placenta, resulting in a homogenate; C. centrifuging saidhomogenate in order to free the supernatant of cellular debris; d.purifying differentiation associated molecular weight fractions; and e.administering said fractions to a patient in need thereof.
 24. The cellfree extract of claim 23 wherein the differentiation-inducing fractionpurified is of molecular weight of 10-10,000 Daltons.
 25. The cell freeextract of claim 23 wherein the differentiation-inducing fractionpurified is of molecular weight of 300-3,000 Daltons.
 26. A method ofstimulating an immune response to cancer cells by administration of thecell free extract of claim 23, together with an appropriateimmunological adjuvant.
 27. A method of prophylactically treating acancer patient by administration of the cell free extract of claim 23 toa patient in need thereof.
 28. A method of inhibiting progression of apreneoplastic lesion to neoplasia by administration of the cell freeextract of claim
 23. 29. A cell free extract capable of inducingdifferentiation in cancer cells, the cell free extract produced by theprocess comprising: a. extracting bone marrow stem cells or unpurifiedbone marrow nucleated cells; b. homogenizing said cells, resulting in ahomogenate; c. centrifuging said homogenate in order to free thesupernatant of cellular debris; d. purifying differentiation associatedmolecular weight fractions; and e. administering said fractions to apatient in need thereof.
 30. A differentiation inducing composition,suitable for administration to a patient in need thereof, derived fromplacental tissue, the composition produced by the process comprising: a.extracting human placenta; b. homogenizing said placenta, resulting in ahomogenate; c. centrifuging said homogenate in order to free thesupernatant of cellular debris; d. purifying the cell free-debristhrough adsorption on a C-18 column; and e. filter sterilizing saidcomposition.
 31. The composition of claim 30, wherein the homogenizedportion of the placenta consists substantially of chorionic tissue. 32.The composition of claim 30, wherein a histone deacetylase inhibitor isadded directly to the composition.
 33. A method of inducingdifferentiation and reprogramming of distal cancer lesions through anintratumoral administration of the composition described in claim 30.34. A method of substantially increasing efficacy of the composition ofclaim 30 by administration in a liposomal complex.
 35. A differentiationinducing composition, comprising a substantially purified therapeuticcomposition prepared through the following steps: (a) preparing asupernatant; (b) subjecting the supernatant to solid phase extraction toproduce a product; (c) subjecting the product from step (b) to gelfiltration to produce a product; (d) subjecting the product from step(c) to anion exchange fast phase liquid chromatography to produce aproduct; (e) subjecting the product from step (d) to amino-highperformance liquid chromatography to produce a product; and (f)subjecting the product from step (e) to reverse phase high performanceliquid chromatography to produce purified differentiation inducingfactor.
 36. The composition of claim 35, wherein said supernatant isprepared by culturing cord-blood derived stem cells to produce afactor-containing supernatant.
 37. The composition of claim 35, whereinsaid supernatant is prepared through centrifugation of lysed placentalextract.